toxins Article Gliotoxin Aggravates Experimental Autoimmune Encephalomyelitis by Triggering Neuroinflammation Thais Fernanda de Campos Fraga-Silva 1,* , Luiza Ayumi Nishiyama Mimura 1, Laysla de Campos Toledo Leite 2, Patrícia Aparecida Borim 3, Larissa Lumi Watanabe Ishikawa 1 , James Venturini 3, Maria Sueli Parreira de Arruda 2 and Alexandrina Sartori 1 1 Department of Microbiology and Immunology, Institute of Biosciences of Botucatu, São Paulo State University (UNESP), Botucatu 18618-691, São Paulo, Brazil 2 Department of Biological Sciences, School of Sciences, São Paulo State University (UNESP), Bauru 17033-360, São Paulo, Brazil 3 Department of Tropical Diseases and Image Diagnosis, Botucatu Medical School, São Paulo State University (UNESP), Botucatu 18618-687, São Paulo, Brazil * Correspondence: [email protected]; Tel.: +55-14-3880-0415 Received: 24 June 2019; Accepted: 23 July 2019; Published: 26 July 2019 Abstract: Gliotoxin (GTX) is the major and the most potent mycotoxin that is secreted by Aspergillus fumigatus, which is capable of injuring and killing microglial cells, astrocytes, and oligodendrocytes. During the last years, studies with patients and experimental models of multiple sclerosis (MS), which is an autoimmune disease of the central nervous system (CNS), suggested that fungal infections are among the possible initiators or aggravators of this pathology. The deleterious effect can occur through a direct interaction of the fungus with the CNS or by the toxin release from a non-neurological site. In the present work, we investigated the effect of GTX on experimental autoimmune encephalomyelitis (EAE) development. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein and then intraperitoneally injected with three doses of GTX (1 mg/kg b.w., each) on days 4, 7, and 10. GTX aggravated clinical symptoms of the disease in a dose-dependent way and this outcome was concomitant with an increased neuroinflammation. CNS analyses revealed that GTX locally increased the relative expression of inflammatory genes and the cytokine production. Our results indicate that GTX administered in a non-neuronal site was able to increase neuroinflammation in EAE. Other mycotoxins could also be deleterious to many neurological diseases by similar mechanisms. Keywords: multiple sclerosis; experimental autoimmune encephalomyelitis; mycotoxin; gliotoxin; immunomodulation Key Contribution: Correlation between fungal infections and CNS neurodegenerative diseases is reinforced by the aggravation of experimental model of MS triggered by gliotoxin. This mycotoxin was able to induce inflammation and demyelination at the CNS even when administered in a non-neuronal site. 1. Introduction Gliotoxin (GTX) is a mycotoxin that was originally isolated from Gliocladium culture. However, it has been lately described that it could also be produced by other fungal species, e.g., Aspergillus fumigatus, Eurotium chevalieri, Trichoderma virens, Neosartorya pseudofischeri, and some Penicillium and Acremonium species [1]. GTX is considered a virulence factor; it intensifies fungal invasiveness [2] and reduces the specific immunity against the fungus [3]. Even though the denomination “gliotoxin” is being Toxins 2019, 11, 443; doi:10.3390/toxins11080443 www.mdpi.com/journal/toxins Toxins 2019, 11, 443 2 of 12 applied to both mycotoxins [4] and chemical compounds toxic for glial cells, as 3-chloropropanediol [5], the possible effects of fungi-derived GTX as triggers or aggravators of inflammatory central nervous system (CNS) disorders have not been comprehensively investigated so far. CNS fungal infections are associated with considerable morbidity and mortality and they comprise a wide spectrum of clinical syndromes, including abscesses, meningitis, meningoencephalitis, stroke, vasculitis, and spinal pathologies, such as arachnoiditis [6]. The main etiological agents of these infections are Aspergillus, Cryptococcus, Candida, Mucorales, dematiaceous molds, and dimorphic endemic fungi. The primary routes of infection are respiratory or traumatic inoculation with subsequent hematogenous or contiguous spreading [7]. Certain fungal species that are secluded in non-neuronal tissues can release toxins which can then reach distant tissues, including the CNS, and, in this case, destroy astrocytes and oligodendrocytes [8], which possibly contributes to exacerbate CNS inflammation. Multiple sclerosis (MS) is an autoimmune disease that is characterized by the presence of autoreactive T cells specific for CNS antigens. Environmental risk factors that are considered to be essential for MS development include infectious and non-infectious factors, as, for example, diet composition, cigarette smoking, and intensity of sunlight exposure [9,10]. Most chronic inflammatory CNS disorders have an infectious origin, and a strong association of viral and bacterial infections with MS was already described [11–13]. More recently, Benito-León and Laurence reviewed the possible role of fungal infections in MS development [14]. Several aspects of MS immunopathogenesis, aggravating factors, and therapy approaches have been addressed in experimental models of the disease, such as the experimental autoimmune encephalomyelitis (EAE). The prototypical model is mainly induced in mice by immunization with neuronal antigens emulsified with Freund’s complete adjuvant. In the present work, we investigated the effect of GTX in EAE development. This GTX was derived from Gliocladium fimbriatum and it was administered by intraperitoneal route, i.e., away from the CNS. This approach allowed us to evaluate the ability of GTX, that was administered in a non-neuronal site, to induce inflammation and demyelination in the CNS. 2. Results 2.1. Gliotoxin Aggravates EAE Evolution C57BL/6 mice were injected with this toxin at the fourth, seventh, and tenth days after EAE induction (EAE/GTX group) to evaluate the potential of GTX to affect encephalomyelitis development, as indicated by arrows in Figure1A. The evaluation of the clinical score revealed that the disease onset, which was clinically characterized by paralysis appearance, started between the 9th and 10th days in both EAE control (EAE) and EAE/GTX groups. From the 14th day of the disease on, the EAE/GTX group displayed more pronounced clinical signs in comparison to the EAE group, including a higher clinical score (Figure1A) and more accentuated body weight loss (Figure1B). Seventeen days after EAE induction, the EAE/GTX group presented a significantly higher maximum score, which corresponds to the mean of the highest degree of paralysis that was reached by all animals (Figure1C), associated with a striking loss of body weight (Figure1D). GTX per se neither affected the survival curve nor the liver and kidney function parameters. Contrarily, the injection of GTX in EAE mice caused two deaths in a group with 12 mice, therefore decreasing the animal survival to 83% (Figure1E). Regarding the liver and kidney function, GTX injection in normal mice (GTX group) did not affect the evaluated clinical biochemical parameters. However, EAE development per se already triggered a reduction in the hepatic enzymes, as shown for ALT (Figure1F), AST (Figure1G), and Alk phos (Figure1H). Urea (Figure1I) and creatinine (Figure1J) were also below normal levels. Some of the alterations that were associated with EAE seemed to be reverted by GTX, such as AST, ALT, and creatinine levels. Toxins 2019, 11, 443 3 of 12 Toxins 2019, 11, x FOR PEER REVIEW 3 of 13 2.2. 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CNS Although(Figure 2C), the mildonly the dose higher (0.5 mg dose/kg) of increased GTX significantly the total number increased of leucocytesIL-17 (Figure in the 2D) CNS and(Figure IL-2 (Figure2C), only2E) theproduction higher dose by CNS of GTX cell significantly cultures that increased were stimulated IL-17 (Figure with2D) the and neuroantigen IL-2 (Figure 2(MOG).E) production We next byevaluated CNS cell possible cultures thatimmunological were stimulated mechanisms with the involved neuroantigen in this (MOG). process We while next evaluatedtaking into possible account immunologicalthat the highest mechanisms dose of GTX involved was able in to this exacer processbate while the clinical taking encephalomyelitis into account that the development. highest dose of GTX was able to exacerbate the clinical encephalomyelitis development. Figure 1. GTX aggravates encephalomyelitis (EAE) development. Normal and EAE mice received threeFigure GTX 1. dosesGTX (1.0aggravates mg/kg). encephalomyelitis Clinical score (A) and (EAE) body deve weightlopment. (B) were Normal daily analyzedand EAE until mice the received acute diseasethree GTX phase doses (day 17).(1.0 Maximummg/kg). Clinical clinical score score ( (AC)), and percentage body weight of weight (B) variationwere daily (D analyzed), and percentage until the ofacute